Kinetics of enzyme acylation and deacylation in the penicillin
acylase-catalyzed synthesis of b-lactam antibiotics
Wynand B. L. Alkema, Erik de Vries, Rene
´
Floris and Dick B. Janssen
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen,
the Netherlands
Penicillin acylase catalyses the hydrolysis and synthesis of
semisynthetic b-lactam antibiotics via formation of a cova-
lent acyl-enzyme intermediate. The kinetic and mechanistic
aspects of these reactions were studied. Stopped-flow
experiments with the penicillin and ampicillin analogues
2-nitro-5-phenylacetoxy-benzoic acid (NIPAOB) and
D
-2-nitro-5-[(phenylglycyl)amino]-benzoic acid (NIPGB)
showed that the rate-limiting step in the conversion of
penicillin G and ampicillin is the formation of the acyl-
enzyme. The phenylacetyl- and phenylglycyl-enzymes are
hydrolysed with rate constants of at least 1000 s
)1
and
75 s
)1
, respectively. A normal solvent deuterium kinetic
isotope effect (KIE) of 2 on the hydrolysis of 2-nitro-5-
[(phenylacetyl)amino]-benzoic acid (NIPAB), NIPGB and
NIPAOB indicated that the formation of the acyl-enzyme
proceeds via a general acid–base mechanism. In agreement
with such a mechanism, the proton inventory of the k
cat
for

Penicillin acylase (PA) (EC 3.5.1.11) of Escherichia coli
ATCC 11105 hydrolyses penicillin G to produce phenyl-
acetic acid and 6-aminopenicillanic acid (6-APA). The latter
compound can be used industrially as a precursor for the
synthesis of semisynthetic b-lactam antibiotics. In synthetic
reactions, 6-APA is coupled to an acyl group in a kinetically
controlled conversion in which the acyl group is supplied as
an activated precursor, e.g. an ester or an amide [1]. This
condensation reaction can also be catalysed by PA and the
yield of the desired product is strongly dependent on the
mechanism and kinetic properties of the catalyst [2].
The catalytic mechanism of PA involves nucleophilic
attack of the active-site serine, bS1, on the carbonyl carbon
of the amide or ester bond of the substrate [3] (residues are
labelled to indicate the subunit (a or b) and their position in
the subunit). Via a tetrahedral intermediate that is stabilized
by hydrogen bonds between the oxyanion hole residues
bN241 and bA69 and the negatively charged oxygen of
the substrate, an acyl-enzyme is formed concomitant with
expulsion of the leaving group [3–5]. The acyl-enzyme may
be deacylated by H
2
O, yielding the hydrolysis product and
thefreeenzyme.Inthismechanism,thefreeN-terminal
NH
2
group of the catalytic serine functions as the catalytic
base, activating the nucleophile by proton abstraction in the
acylation reaction. In the presence of a b-lactam nucleo-
phile, such as 6-APA or 7-aminodesacetoxycephalosporanic

-phenylglycine-p-nitroanilide;
pNPA, p-nitrophenylacetate; KIE, kinetic isotope effect.
Enzymes: penicillin acylase (PA; EC 3.5.1.11).
(Received 3 April 2003, revised 13 June 2003, accepted 26 June 2003)
Eur. J. Biochem. 270, 3675–3683 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03728.x
expression for yield of the desired product, i.e. the maximum
product accumulation [Q]
max
[8]:
[Q]
max
¼
K
Q
k
hQ
Á
k
2
K
AD
Á
[N]Ák
s
k
h1
ÁK
EAcN
þ k
h2

Materials and methods
Kinetic measurements
All kinetic experiments were done with purified penicillin
acylase of E. coli ATCC 11105, which was obtained as
described previously [4]. The enzyme concentration was
determined by measuring A
280
and using e ¼
210 000
M
)1
Æcm
)1
. The conversion of the chromogenic
substrates 2-nitro-5-[(phenylacetyl)amino]-benzoic acid
(NIPAB) (De
405nm
¼ 9.09 m
M
)1
.cm
)1
), phenylacetyl-p-
nitroanilide (PAPNA) (De
405nm
¼ 13 m
M
)1
Æcm
)1

) was followed by meas-
uring the increase in absorbance change at 405 nm, on a
Perkin Elmer Bio40 UV/VIS spectrophotometer at 30 °Cin
50m
M
phosphate buffer, pH 7.0. The stopped-flow experi-
ments were carried out on an Applied Photophysics
SX.17MV spectrophotometer, with a 10-mm optical path
length. The stopped-flow cell was thermostatically con-
trolled at 30 °C. Stock solutions of all ester substrates were
made in acetonitrile and diluted to the appropriate concen-
tration in 0.5% acetonitrile in 50 m
M
phosphate buffer,
pH 7.0, prior to the start of kinetic experiments. Conversion
of non chromogenic substrates was followed using HPLC as
described [4]. Nucleophile competition experiments were
carried out by mixing enzyme with solutions of acyl donor
and nucleophile. The enzymatic conversions were followed
by HPLC and the V
s
/V
h
ratios were calculated from the
initial rates of the formation of synthesis and hydrolysis
products.
Buffers for experiments in D
2
O were prepared as follows:
solutions of 50 m

O, pH 7.0.
The values for De were corrected for the influence of D
2
Oon
the extinction coefficients, which was measured with all
H
2
O/D
2
O mixtures that were used. Proton inventories were
carried out at pL 7.0, which is in the pH-independent region
for PA catalysed hydrolysis reactions [9].
Fitting of kinetic parameters to the data was done using
the program
SCIENTIST
(Micromath Inc., version 2.0). The
goodness of fit and the information content of the data were
checked by inspecting the value for the model selection
criterion, standard deviations for the 95% confidence
interval of the parameter values and the correlation matrix
for the fitted parameters [16]. These parameters were
calculated using the Statistics procedure implemented in
the
SCIENTIST
program.
Chemicals
NIPAB, p-NPA, phenylacetic methyl ester and phenylacetic
acid ethyl ester were purchased from Sigma Chemical
Company. NIPAOB, NIPGB and 5-(2-amino-2-phenyl-
acetoxy)-2-nitro-benzoic acid (NIPGBester) were purchased

Q
, K
AD
and K
N
,
K
EAcN
are the binding constants of the substrates to the free enzyme
and the acyl-enzyme, respectively.
3676 W. B. L. Alkema et al.(Eur. J. Biochem. 270) Ó FEBS 2003
HCl and 1
M
NaOH. After drying and evaporation a white-
yellow solid was obtained that was recrystallised from
methanol/ether. Mp. 114–115 °C (uncorr.).
1
HNMR
(300 MHz, dimethylsulfoxide d6) d (p.p.m.): 3.71 (s, 2H,
CH
2
); 7.26–7.35 (m, 5H, CH); 7.82 (d, J ¼ 9.0 Hz, 2H,
CH); 8.20 (d, J ¼ 9.0, 2H, CH); 10.77 (brs, 1H, NH).
Phenylacetamide was prepared as follows: phenylacetyl-
chloride was added dropwise to concentrated ammonia
solution. This gave the product as a white precipitate that
was filtered off and dried to constant weight. Mp. 152–
153 °C (uncorr.).
1
H NMR (300 MHz, D

¼
k
2
Ák
h1
k
2
þ k
h1
ð3Þ
and
K
m
¼
K
AD
Ák
h1
k
2
þ k
h1
ð4Þ
If deacylation of the enzyme is much slower than
acylation, i.e. k
h1
( k
2
, the k
cat

values for the methyl and
ethyl ester of phenylacetic acid were approximately fivefold
higher than for the best amide substrate, phenylacetamide,
which is in agreement with the fact that amide bonds are in
general more stable than ester bonds. The k
cat
/K
m
for
NIPAOB was more than threefold higher than for penicillin
G, which makes NIPAOB the best substrate known so far
for penicillin acylase. The k
cat
values for phenylacetic acid
methyl ester and phenylacetic acid ethyl ester were only
slightly lower than the k
cat
of NIPAOB, indicating that
increasing the reactivity of the carbonyl function of the
substrate by using p-hydroxynitrobenzoic acid as the leaving
group, did not lead to a higher rate of conversion. For esters
of phenylglycine the k
cat
was similar to the k
cat
for
phenylglycine amide, indicating that the higher reactivity
of the ester bond also did not lead to an increased rate of
conversion.
These results indicate that for hydrolysis of the phenyl-

calculated [18,19]. To obtain the values for k
2
and k
h1
,we
performed stopped-flow experiments with the chromogenic
Table 1. Steady-state parameters of penicillin acylase for the hydrolysis
of esters and amides of phenylacetic acid and phenylglycine. The struc-
tures of the chromogenic substrates are shown in Fig. 2. The reaction
conditions are given in the Materials and methods section. Values
represent means of three experiments. The standard deviation was in
all cases within 10% of the mean values.
Substrate
k
cat
(s
)1
)
K
m
(m
M
)
k
cat
/K
m
(m
M
Æs

slower than the rate of deacylation. The theoretical maxi-
mum of the amplitude of the burst phase is equal to the total
enzyme concentration. When k
h1
¼ 5 k
2
, only 2% of the
maximum of the amplitude of the burst phase can be
observed which is considered to be the lower limit of
detection. Since the k
cat
for the hydrolysis of NIPAOB is
200 s
)1
, hydrolysis of the acyl-enzyme must take place at a
rate of at least 1000 s
)1
. Likewise, the lower limit for the
hydrolysis of the phenylglycylated enzyme is at least 75 s
)1
,
which is 5 k
cat
for the hydrolysis of NIPGB. Attempts to
obtain a more accurate estimate for the lower limit of k
h1
,by
using the more reactive ester of NIPGB in kinetic experi-
ments, failed because of the high rates of spontaneous
hydrolysis of this compound.

2
OontheK
m
was observed. For the substrates NIPAB
and NIPAOB, a proton inventory was recorded by
measuring the k
cat
in mixtures of H
2
OandD
2
O (Fig. 4).
The relation between the value of a rate constant and the
mole fraction D
2
O can be described by the simplified form
of the Gross–Butler equation [20],
k
x
k
0
¼
Y
v
i
ð1 À x þ x/
T
i
Þð5Þ
In this equation k

for NIPAB, suggesting that one proton is transferred in
the transition state of the reaction. Fitting to the Gross–
Butler equation yielded a fractionation factor of 0.5 for the
proton that is exchanged. This fractionation factor is
indicative of general acid/base catalysis mechanism where
the proton is less tightly bound in the transition state than in
the reactant state [21], in agreement with the mechanism of
PA-catalysed hydrolysis as proposed by Duggleby et al.
(1995). The proton that gives rise to the normal isotope
effect may be the proton that is transferred from the seryl
oxygen to the seryl amino group during activation of the
nucelophilic serine, or the proton that is donated to the
leaving group during collapse of the tetrahedral intermediate.
Fig. 3. Stopped-flow traces using NIPGB, NIPAOB and p-NPA. For
NIPGB and NIPAOB, the final enzyme concentration was 4 l
M
and
substrate concentration was 10ÆK
m
. For p-NPA the final enzyme
concentration was 3 l
M
and substrate concentration was 400 l
M
.
Fig. 4. Effect of D
2
O and glycerol on the hydrolysis of NIPAB and NIPAOB. (A) Proton inventories for k
cat
of NIPAB and NIPAOB. k

O, whereas there was an almost linear
decrease of the k
cat
at larger mole fractions of D
2
O. Unlike
with NIPAB, these data could not be fitted to Eqn (5), or to
a modified Gross–Butler equation taking medium effects
into account [22]. The higher viscosity of D
2
O compared to
H
2
O and the isotope effects on H-bonds have been described
to influence the enzyme structure, flexibility and stability
[23–27]. D
2
O effects on rate constants that result from
changes in solvent viscosity instead of proton transfer in the
rate-limiting step have been described for chymotrypsin,
NAD-malic enzyme and alkaline phosphatase [28–30]. It is
conceivable that the increase in activity of PA may be caused
by small structural changes in the active site at low
concentrations of D
2
O, making enzyme–substrate inter-
actions more favourable for catalysis than in H
2
O.
To study the influence of solvent viscosity on the

0
¼
K
x
þ k
0
Áx
K
x
þ x

Á
Y
v
i
ð1 À x À x/
T
i
Þð6Þ
The second term is the simplified form of the Gross-
Butler equation and describes the decrease in rate due to
proton transfer in the transition state. Fitting Eqn (6) to
these data yielded values of 1.58 ± 0.03 for k¢ and
0.0078 ± 0.0016 for K
x
. The best fit was obtained
assuming a two-proton transfer mechanism, suggesting
that two protons are transferred in the transition state of
this reaction. However, a distinction between a one-
proton and a multiple-proton mechanism can only be

experiments [7]. The rate of aminolysis vs. hydrolysis can be
expressed as the V
s
/V
h
ratio and according to Scheme 1, is
given by [33]:
V
s
V
h
¼
[N]Ák
s
k
h1
ÁK
EAcN
þ k
h2
Á½N
ð7Þ
The above kinetic experiments showed that the hydro-
lysis of the phenylacetylated enzyme (k
h1
) proceeds with
a rate of at least 1000 s
)1
. To obtain significant rates of
aminolysis at such a high rate of hydrolysis, it follows

s
/V
h
vs.
[N] is hyperbolic and may be written as
V
s
V
h
¼
V
s
V
h

max
Á½N
K
Napp
þ½N
ð8Þ
in which
V
s
V
h

max
¼
k

EAcN
) and the ratio between the rates of hydrolysis of
the acyl-enzyme with nucleophile bound (k
h2
) and
without nucleophile bound (k
h1
). In other words, when
binding of the b-lactam nucleophile leads to a reduction
of the rate of deacylation by H
2
O by lowering the
nucleophilicity of H
2
O or by displacement of water from
the active site, the apparent affinity for the b-lactam
nucleophile decreases (K
Napp
increases) and the relation-
ship between V
s
/V
h
and [N] will approach to a straight
line, given by,
V
s
V
h
¼

and the K
Napp
for this
reaction were higher than observed for synthesis of
ampicillin.
Values of 47 ± 8 m
M
for K
Napp
and of 3.6 ± 0.22 for
(V
s
/V
h
)
max
for the synthesis of ampicillin were obtained
from fitting Eqn (8) to the data. For synthesis of penicillin
G, a value of 0.018 m
M
)1
for the slope of the curve was
obtained, but due to the almost linear dependence of V
s
/V
h
on the concentration of 6-APA no reliable values for
(V
s
/V

the saturation at a lower V
s
/V
h
value that was found with
6-APA as the nucleophile, indicating that the enzyme has a
lower apparent affinity for 7-ADCA (Fig. 5B).
Yousko et al. (2002) found similar constants for the
V
s
/V
h
for deacylation with 7-ADCA and 6-APA using PAA
and PGA. However they observed in all cases a saturation
of the (V
s
/V
h
)
max
, whereas our data indicate a linear relation
for the combinations of 7-ADCA/PGA and 6-APA/PAA.
Since their measurements were carried out at a different
pH, this indicates that competition between water and
the nucleophilic b-lactam at the active-site may be
pH-dependent.
From Eqn (10) it follows that the higher K
Napp
for
7-ADCA may be caused by a lower affinity of the enzyme

Fig. 5. Nucleophile competition experiments with penicillin acylase.
(A) Dependence of V
s
/V
h
on [6-APA], using phenylacetamide (PAA)
or phenylglycine amide (PGA) as the acyl donor. (B) Dependence of
V
s
/V
h
on [N], using 7-ADCA or 6-APA as the nucleophiles and
phenylglycine amide as the acyl donor. (C) Dependence of V
s
/V
h
on
[APA], using phenylacetamide as the acyl donor in H
2
O and 100%
D
2
O. The symbols represent experimental data, the lines are the best fit
to the data using Eqn (8).
3680 W. B. L. Alkema et al.(Eur. J. Biochem. 270) Ó FEBS 2003
for K
EAcN
in Eqn (10) revealed that the reduced apparent
affinity for 7-ADCA is not caused by weaker binding of
7-ADCA but by a lower k

amino function exert a large influence on the competition of
the nucleophiles with H
2
O for the acyl-enzyme. The 6-APA
moiety of penicillin G binds with the thiazolidine ring to the
enzyme via hydrophobic interactions between the 2b-methyl
group and aF146 and bF71 and hydrogen bonding between
its carboxylate group and aR145:NH
2
[4,9,34]. In 7-ADCA,
which has a dihydrothiazine ring instead of a thiazolidine
ring, the 2b-methyl group is not present and due to the more
planar character of the dihydrothiazine ring, the carboxylate
group may be in a different position than in 6-APA.
Consequently, it is conceivable that binding of 7-ADCA to
the enzyme is mediated via different interactions than
binding of 6-APA, which may explain the higher affinity of
the enzyme for 7-ADCA and the higher nucleophilicity of
7-ADCA compared to 6-APA.
Proton transfer in the deacylation reaction
To study whether proton transfer would be important in the
deacylation of phenylacetylated acyl-enzyme, we measured
the initial V
s
/V
h
in D
2
O at several concentrations of 6-APA
(Fig. 5C). The V

transition states of the deacylation reaction or from separate
effects of D
2
O on the transition states of both the hydrolysis
and aminolysis reaction. The latter possibility seems more
likely, in view of the one-proton transfer mechanism
suggested for deacylation of PA [3,35].
Conversion of p-NPA leads to an acyl-enzyme in which
an acetyl group is attached to the active-site serine.
Deacylation of this acetyl-enzyme is the rate-limiting step
in the catalytic cycle. No solvent KIE was observed on the
k
cat
of p-NPA suggesting that in the transition state of this
hydrolytic reaction no protons are transferred (Fig. 7). This
indicates that in this deacylation reaction a chemical
reaction involving proton transfer is not the rate-limiting
step, in contrast to deacylation of the phenylacetylated
enzyme. The rate constant for deacylation of the acetyl-
enzyme may reflect another step, such as a conformational
change of the enzyme prior to chemical hydrolysis of the
acyl-enzyme by water. A conformational change in the
deacylation of p-NPA is not unlikely in view of structural
results obtained by Done et al.[36].Inthisworkitwas
shown that p-nitrophenylacetic acid may bind in the acyl-
binding site of PA. Hydrolysis of p-NPA may thus proceed
via reversed binding of the substrate, in which the leaving
group, p-nitrophenol, occupies the acyl binding site and the
acetyl group binds at the leaving group binding site,
sterically hindering the deacylating H

hydrolysis of p-NPA on the molar fraction D
2
O.Forthissubstrate,
acylation is faster than deacylation and the k
cat
thus represents the rate
of breakdown of the acyl-enzyme intermediate.
Ó FEBS 2003 Kinetic analysis of penicillin acylase (Eur. J. Biochem. 270) 3681
Structural analysis of an enzyme-substrate complex could
confirm the existence of such a reversed binding mode [4,5].
Kinetic constants for PA catalyzed synthesis
and hydrolysis of b-lactam antibiotics
The kinetic properties of PA are important with respect to
the yield that can be obtained in a kinetically controlled
synthesis of b-lactam antibiotics. Combining the data from
the steady-state and presteady state experiments and the
data from nucleophile competition and inhibition experi-
ments, the kinetic constants for the synthesis and hydrolysis
of b-lactam antibiotics can be calculated (Table 2). Exact
values can be determined for the rate constants of acylation,
whereas only relative rates can be obtained for the deacy-
lation by various nucleophiles. The values for the hydrolysis
and synthesis of ampicillin are close to the numbers
obtained by Yousko and Svedas [15]. Eqn (1) shows that
for the application of the enzyme in synthesis two kinetic
properties are important. First, the enzyme should have a
low activity for the antibiotic compared to the acyl donor.
The relative specificity of the enzyme for both substrates
may be expressed by the factor a, given by [7],
a ¼

[37,38]. Furthermore the K
m
for the product ampicillin
is much lower than the K
m
values for the acyl donors
phenylglycine amide and phenylglycine methyl ester.
Both the low activity of PA for ester substrates
compared to amides and the high affinity of the enzyme
for the product of synthesis increase a and hence reduce
the yield in synthesis reactions.
A second requirement for efficient synthesis is a high rate
of aminolysis compared to hydrolysis. The experiments with
the most important nucleophiles in b-lactam antibiotic
synthesis, 7-ADCA and 6-APA, showed that tight binding
of the nucleophile to the acyl-enzyme and displacement of
the hydrolytic water molecule are the two most important
factors in determining the reactivity of the nucleophile. The
fact that the acyl-enzyme to which 7-ADCA is bound
cannot be hydrolysed, signifies that the V
s
/V
h
ratioina
synthesis reaction can be increased by adding more
7-ADCA. In contrast, when using 6-APA as the nucleo-
phile, the V
s
/V
h

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the k
2
to the value obtained for the k
cat

) 1.2 2.5 0.005
k
hQ
(s
)1
)50 30 40
k
h1
(s
)1
) >75 >75 >1000
k
h2
(s
)1
)<k
s
/35 k
s
/3 <k
s
/4
k
s
(s
)1
) >308 >150 >4000
K
EacN
(m

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